The present invention is generally related to liquid handling systems for dispensing and aspirating small volumes of liquids of the order of 1 ml and smaller, 100 microliters and smaller, or even 100 nl or smaller. Liquid handling systems are being provided and will be provided in the future for the dispensation of droplets of even smaller volumes.
The present invention is particularly directed to liquid handling systems used in life science, medical and pharmaceutical sectors for applications such as high throughput screening, microarraying, Polymerase Chain Reaction (PCR), combinatorial chemistry, proteomics, protein crystallography, genetic screening and others. The invention may also be used for medical diagnostics e.g. for printing reagents on a substrate covered with bodily fluids or for printing bodily fluids on substrates.
The development of instrumentation for dispensing minute volumes of liquids has been an important area of technological progress for some time. Numerous devices for dispensing of small volumes of liquids of the order of 100 microliters and smaller have been developed over the past twenty-five years.
The requirements of a dispensing system vary significantly depending on the application. For example the main requirement of a dispensing system for ink jet applications is to deliver a droplet of a fixed volume with a high repetition rate. The separation between individual nozzles making up the ink jet dispenser should be as small as possible so that a number of nozzles may be accommodated on a single printing cartridge. For this particular end use, the task is simplified by the fact that the properties of the liquid dispensed, namely ink, are well defined and consistent.
On the contrary, for life science, medical and pharmaceutical applications the requirements are completely different. The system should be capable of handling a variety of liquids with different mechanical and physical properties, e.g. viscosity. For many such applications it is important to be able to freely adjust the volume dispensed. Recent inventions related to the field of small volume liquid handling are covered by numerous patent applications, e.g. U.S. Pat. No. 5,744,099 (Chase et al); U.S. Pat. No. 4,574,850 (Davis); U.S. Pat. No. 5,035,150 (Tomkins), U.S. Pat. No. 5,741,554 (Tisone); U.S. Pat. No. 6,713,021 (Shvets), U.S. Pat. No. 6,669,909 (Shvets) and are also described in the following scientific and technical publications:    J. Comley, Nanoliter Dispensing—on the Point of Delivery, Drug Discovery World, Summer 2002, p 33-44;    D. Rose, Microdispensing Technologies in Drug Discovery, Drug Discovery Technology, vol 4, N9, September 1999, p 411-419;    S. D. Rose, Applications of a Novel Microarraying System in Genomics Research and Drug Discovery, Journal of the Association for Laboratory Automation, vol 3, N3, 1998, p 53-56;    I. V. Shvets, S. Makarov, C. Franken, A. Shvets, D. Sweney, J. Osing, Spot on Technology of low volume liquid handling, Journal of Association of Laboratory Automation, vol 7, N 6, December 2002, p 127-131.
The wide variety of the mechanical properties of liquids and the very nature of many biological liquids often make consistent dispensing difficult. For example the dispenser can be easily blocked by a clot as these are readily formed in cell-based liquids and protein solutions. Therefore, it is highly desirable to be able to verify that the instrument is dispensing correctly and in many cases to be able to detect the moment when the dispenser runs out of the liquid or starts malfunctioning. It is also desirable to be able to detect if the liquid handling instrument leaks or if a drop fails to separate from the dispensing nozzle of the instrument. Additionally, in many instances, it is advantageous to be able to measure the volume of the droplet dispensed to ensure that it does correspond to the amount requested by the operator of the dispensing system. Independent verification of the volume dispensed becomes more and more important as the users have to operate in an environment of increasing legal regulation. For example, failure to dispense a drop may lead to an incorrect result of a medical test and consequently to incorrect diagnosis. Therefore, the user requirement for availability of such measurement technologies for operation verification intensifies.
It is also desirable for manufacturers of various liquid handling instruments to have in-house instrument test and calibration tools. During the production phase and also during the phase of development of new liquid handling instruments, it is important to have tools for quality control, calibration, tuning and optimisation of the instruments.
The issue of droplet volume measurement for small volume dispensing is also important from a psychological perspective. The reason is that in many cases the operator cannot readily monitor visually arrival of a tiny drop to a destination substrate, in particular, if the destination substrate is not flat. This adds to the user discomfort even if the dispenser functions properly as the user cannot readily satisfy this by simple visual monitoring.
It is difficult to fulfil challenging requirements for a successful system for measurement of droplet volume during dispensing. For many applications the ideal system must measure the volume in non-contact mode meaning that direct transfer of the drop to a measurement device, such as microbalance, is not an option.
U.S. Pat. No. 5,559,339 (Domanik) teaches a method for verifying a dispensing of a liquid droplet of relatively large size from a nozzle. The method is based on coupling of light from a source to a receiver. As a droplet of liquid travels from the nozzle it obstructs the coupling and therefore, the intensity of the signal detected by the receiver is reduced. The disadvantage of this method is that it is based on the absorption of electromagnetic radiation (in practice light) by the droplet. For a range of applications where minute droplets of liquids with a broad range of optical properties need to be dispensed may present a challenge. Another limitation of U.S. Pat. No. 5,559,339 (Domanik) is that for many applications it is desirable not only to confirm that a droplet has been dispensed but also extract information on the size of the droplet, and its velocity and shape. U.S. Pat. No. 5,559,339 did not focus on these issues.
There are many inventions dealing with the control of the dispensing for the ink jet printing applications. It should be noted that in the ink jet printing application the range of the droplet volumes is entirely different to the range of interest in the present application. In ink jet printing application the droplets often have the volume in the pico-liter range. This is some 100 times smaller than the typical droplets dealt with in the present invention which are normally in the range from 10 nl to 50-1000 microliters. Therefore many approaches developed for the applications in ink jet printing are not suitable for the field of dispensing for life science and drug discovery.
Furthermore, many inventions in the ink-jet field are directed to monitoring the passage of the droplet amounts to simple observation of the signal intensity reduction on the detector or measuring time lag between two signals from the two adjacent detectors. These inventions usually do not teach how more complex analysis of the droplet could be obtained from the measurements.
There is another substantial difference between the inventions related to the ink jet printing and the one from the field of use of the present invention. For droplets as small as used in ink jet printing, the volume of the droplet is related to its size via a simple relationship: the volume is proportional to the third power of its size. This is merely a reflection of the fact that the droplet has a sphere-like shape. The greater is the size of the droplet, the greater is the absorption of light passing through it. This simple relationship is the basis of the volume measurements methods in some of the above patents. In contrast, as it will be clear from the description below, in the droplet volume range that is of interest for the field of use of the present invention (for example, 1000 μl or less, preferably 1 nl to 1000 μl, 10 nl to 100 μl, and/or 5 nl to 50 μl), this relationship generally is not valid. Therefore, the methods described for other applications (ink-jet, etc.) could not be applied in the present invention.
For measurement of the droplet volume for the field of use of the present invention, i.e. low volume liquid handling for life science and medical fields, the most common method used is based on electric measurements. The detection relies on measurements of a charge carried by a droplet. The droplets are typically charged by applying a high voltage to the dispenser. The charge carried by the droplet is reflective of the droplet's size. In some inventions it is proposed using a Faraday pail for this purpose (U.S. Pat. No. 6,713,021 (Shvets); European Patent No 1,099,484 (Shvets); U.S. Pat. No. 6,669,909 (Shvets)). Faraday pails are well known and described in many published documents. Essentially the Faraday pail consists of an inner box and a shield. The shield and the box are well insulated from each other. In this situation a charged droplet arriving at the box induces a charge of the opposite sign and same magnitude at the surface of the box. This charge is created by the current flowing to the inner box and it can be measured by a charge measurement circuit. Generally the dispenser and hence the nozzle are maintained at a relatively high voltage. The shield and box are connected to ground potential. The charge can also be measured without catching the droplet in the pail. For this the pail is made in the shape of a cylinder without a bottom. Thus, the charged droplets progress through the Faraday pail that serves as an induced charge detector. The Faraday pail can be used to detect the moment when the droplet enters into the box and leaves it. Therefore, it can be used to measure the droplet's velocity, as the length of the box is known.
The Faraday pail has a number of disadvantages. One of the disadvantages is that it is sensitive to external electromagnetic noise, e.g. noise at 50 or 60 Hz frequency. To measure the volumes of small drops, the sensitivity of the Faraday pail must be optimised meaning that the entry and exit holes of the shield and the inner box must be reduced in size. This increases the chances of missing the exit hole and thus leaving the drop attached to the wall of the inner chamber. Another disadvantage is that in order to increase the charge carried by the droplet and thus improve the sensitivity of the instrument, it is often necessary to apply as high a voltage to the dispenser as possible. This may create further complications; e.g. the risk of destroying a charge sensitive amplifier connected to the pail, the risk of the malfunctioning of high voltage equipment with consequent danger for the operator, etc.
U.S. patent application Ser. No. 10/787,229 filed Feb. 27, 2004 (Shvets et al) describes alternative method of droplet volume detection based on changing the capacitance of the sensor positioned around the nozzle. According to the method, the capacitance between the sensor and the nozzle is measured. Once the droplet is ejected from the nozzle, it increases the capacitance for a short period of time. The volume of the liquid dispensed can be determined from the shape of the electric signal induced in the sensor related to its capacitance change.
The key problem with the many of the droplet volume measurement inventions for the field of low volume liquid handling in life science is the low conductivity of many relevant liquids. Unlike in the case of the ink jet printing where the formulation of the ink could be modified to suit the requirements of the printer, in the area of life sciences it is often not possible to change the formulation of the liquid. Therefore, there is no simple way to increase its conductivity.
U.S. Pat. No. 6,669,909 (Shvets) also describes another method for droplet detection that is very specific to the dispensing apparatus. It is related to monitoring the force acting on the actuator in the dispensing apparatus and also the displacement of the actuator. Unfortunately, this method can only be used with a specific type of the dispensing apparatus.
To summarise, at present the issue of reliable detection and measurement of the volume of droplets dispensed for applications in life science, medical diagnostics, drug discovery and pharmaceutical areas is still not fully resolved. It is suggested that the lack of a commonly acceptable measurement technology impedes wider use of low-volume liquid handling equipment. The same applies to the low volume liquid dispensing for certain other applications outside the field of life science.